The invention relates to the use of quinolone- and naphthyridonecarboxylic acid derivatives which are substituted in the 7-position by a 2-oxa-5,8-diazabicyclo[4.3.0]non-8-yl radical, and their salts for the therapy of Helicobacter pylori infections and the gastroduodenal disorders associated therewith.
With the rediscovery of Helicobacter pylori (H. pylori; formally Campylobacter pylori) by Warren and Marshall in 1983, in the following years it was possible to fundamentally further develop the pathophysiological ideas about the origin of gastroduodenal disorders of man.
H. pylori is regarded as a cause of type B gastritis and appears to play a causal role in the perpetuation of peptic ulcer. Epidemiological and pathological investigations likewise point to a relationship between long-term colonization of the gastric mucosa with the bacterium and the origin of certain forms of carcinoma of the stomach. H. pylori was therefore classified in 1994 as a carcinogen of the first class (most dangerous carcinogenic category). A rare stomach cancer, MALT lymphoma (mucosa-associated lymphoid tissue), likewise often appears to be caused by the bacterium. In initial case reports, after H. pylori eradication not only the reactive infiltrates actually disappeared, but also a part of the poorly malignant MALT lymphoma. Relationships with hypertrophic gastritis are also discussed. The role of H. pylori in functional gastropathy (nonulcerative dyspepsia) is still unclear.
Various epidemiological studies come to the conclusion that about half the world population is infected with the bacterium. The probability of the colonization of the stomach with Helicobacter increases with age. The optimum adaptation of Helicobacter to the living conditions in the unusual, low-competition habitat [lacuna] stomach appears to be the prerequisite for the successful establishment of the chronic infection and for the wide distribution of this pathogenic species.
The pathogenic organisms with their flagella are very mobile not only in the liquid medium, but also in the viscous mucus of the gastric mucous membrane, adhere to the gastric epithelial cells and proliferate best at an oxygen content of 5%, as prevails in the mucus of the gastric wall. Moreover, the bacteria form large amounts of the enzyme urease, which splits urea into ammonia and carbon dioxide. Possibly, the resulting xe2x80x98ammonia cloudxe2x80x99 helps to neutralize the acidic medium in the microenvironment and thus to protect from the aggressive gastric acid.
Peptic Ulcer
The introduction of the histamine H2 receptor antagonists in the 70s was a milestone in the therapy of peptic ulcer. The frequency of surgical interventions for treatment of the ulcer sufferer thus decreased dramatically worldwide. This principle of acid blockage was improved still further by the development of the even more strongly active proton pump inhibitors.
As a result of the antacid therapy, however, only the symptoms of the ulcer, not the natural course of the disorder, which is characterized by the occurrence of relapses, can be influenced causallyxe2x80x94say due to bactericidal treatment. Since virtually all duodenal ulcer patients and the predominant majority of patients with stomach ulcer have an H. pylori infection of the stomach and thus suffer from infectious diseases. Only ulcerations which are caused by non-steroidal anti-inflammatories are not associated with an H. pylori infection.
Therefore, according to the recommendations of a consensus conference, which was organized in 1994 by the American Public Health Authority (NIH), in the case of positive detection of bacteria all patients with peptic ulcers should be subjected to eradication therapy directed against H. pylori (NIH Consensus Statement 1: 1-23; 1994). The arguments were supplied by controlled therapy studies, in which it was possible to show that after successful eradication of bacteria the ulcer recurrence rates fall drastically(0%-29% versus 61%-95%).
H. pylori Therapy
The present eradication of H. pylori turns out to be problematic in practice. A simple and yet reliably effective therapy is not available. The bacterium turns out to be well protected and difficult to attack under the mucous layer.
H. pylori is sensitive in vitro to numerous antibiotics. These are, however, not effective in vivo as a monotherapy. These include, inter alia, penicillin, amoxicillin, tetracyclin, erythromycin, ciprofloxacin, metronidazole and clarithromycin. Bismuth salts and, to a small extent, even proton pump inhibitors (omeprazole, lansoprazole) are antibacterially active in vitro, but not in vivo.
Among all therapy modalities hitherto used for the eradication of H. pylori, at present only the following triple therapies are sufficiently active:
1. classical bismuth triple therapy (bismuth salt plus two antibiotics) and the
2. modified triple therapy (acid inhibitor plus two antibiotics).
However, these regimes are involved eradication procedures with poor compliance, which can be affected up to 35% by side effects (abdominal pain, nausea, diarrhoea, dryness of the mouth, taste disorders and allergic skin reactions, etc.). Wide use is therefore made difficult. A further great disadvantage is the high number of medicaments to be taken daily (12-16 tablets/day). This is particularly marked in the quadruple therapy, in which an acid secretion inhibitor is administered simultaneously to the classical triple therapy.
The better tolerated dual therapy propagated in Germany (combination of amoxicillin with omeprazole) is, however, only poorly effective and appears to fail even largely in patients pretreated with omeprazole and in smokers.
In the triple therapies, the antibiotic components as a rule administered are amoxicillin, nitroimidazole compounds (metronidazole, tinidazole), tetracyclin and recently macrolides (clarithromycin) [in 3-4 sub-doses].
Worldwide, eradication rates of 70-90% are achieved. Various factors can, however, influence this eradication result:
1. In the first place, the resistance of the bacterium (developing countries: up to 60%, Germany: up to 10%) against metronidazole, the most frequently employed antibiotic in triple therapy, can be mentioned. Even in the case of treatment with clarithromycin, the disadvantage of a development of resistance of up to 10% is to be pointed out.
2. As a further factor, the abovementioned compliance of the patients can be mentioned.
Animal Model
An H. felis mouse model has been described as a suitable animal model [A. Lee et al., Gastroentrology 99: 1315-1323 (1990)] and has been modified by us so that it is very highly suitable for the screening and the comparative assessment of abovementioned compounds.
In spite of large morphological differences, the corkscrew-like, urease-forming bacterium H. felis is very closely related to H. pylori. H. felis is a natural inhabitant of the gastric mucosa of dogs and cats. After oral inoculation, the pathogenic bacteria also colonize the mouse stomach in a similar manner to that in which H. pylori colonizes the stomach of man. The established chronic long-term infection leads in mice to active gastritis and induces a corresponding immune response.
The therapeutic effectiveness of test preparations determined in the H. felis mouse model is regarded in the literature as predictive of the corresponding clinical efficacy.
In spite of very good in-vitro activity of antibiotics (e.g. amoxicillin or erythromycin) against H. pylori, after monotherapeutic use clinically these show no significant therapeutic action. This fact is also repeated by the H. felis mouse model. Correspondingly, it was also possible to confirm the clinically recognized eradicative action of the classical triple therapy in the H. felis mouse model.
Antibacterially active 7-(2-oxa-5,8-diazabicyclo[4.3.0]non-8-yl)-quinolone- and naphthyridonecarboxylic acid derivatives have already been disclosed in EP-A-350733 and EP-A-550 903 (Bayer). In JP 8048629 (Dainippon), it was described that compounds such as 8-chloro-1-cyclopropyl-7-([S,S]-2,8-diazabicyclo[4.3.0]non-8-yl)-6-fluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid (BAY Y 3118) have an antibacterial action against H. pylori. It is also known that a number of highly active quinolones, such as, for example, ciprofloxacin, lomefloxacin or ofloxacin (Journal of Antimicrobial Chemotherapy 22, 631-636 [1988], Antimicrobial Agents and Chemotherapy, 33, 108-109 [1989]), have an action against Helicobacter spp. in vitro. It was seen, however, in the animal model (Helicobacter felis, mouse), that these clinically employed antibacterially active quinolones in therapeutically used doses are not able to lead to an eradication of the bacterium. Even by a monotherapeutic treatment with highly active quinolones, which hitherto have not been introduced into the market, such as, for example, with the already mentioned BAY Y 3118, no eradication of H. felis can be achieved in the mouse model without in the main a large part of the animals not dying on account of the toxicity of the compound. The use of trovafloxacin or its derivatives in combination with other antibiotics such as amoxicillin or tetracyclines or proton pump inhibitors such as omeprazole for the therapy of H. pylori is described in EP-676 199 and GB-A-2 289 674 (Pfizer).
The object on which the invention is based was therefore to find relatively highly tolerable active compounds which are able to eradicate this highly specialized bacterium by a simple monotherapy.
It has now been found that compounds of the general formula (I) 
in which
R1 represents alkyl having 1 to 4 C atoms, which is optionally mono- or disubstituted by halogen, phenyl which is optionally substituted by 1 or 2 fluorine atoms or cyclopropyl which is optionally substituted by 1 or 2 fluorine atoms,
R2 represents hydrogen, alkyl having 1 to 4 carbon atoms, which is optionally substituted by hydroxyl, methoxy, amino, methylamino or dimethylamino, or (5-methyl-2-oxo-1,3-dioxol-4-yl)-methyl,
A represents N or Cxe2x80x94R3, where
R3 represents hydrogen, halogen, methyl, methoxy, difluoromethoxy or cyano alternatively, together with R1, can form a bridge of the structure xe2x80x94*Oxe2x80x94CH2xe2x80x94CHxe2x80x94CH3 or xe2x80x94*Oxe2x80x94CH2xe2x80x94Nxe2x80x94CH3, where the atom marked by * is connected to the carbon atom of A,
R4 represents hydrogen, benzyl, C1-C3-alkyl, (5-methyl-2-oxo-1,3-dioxol-4-yl)-methyl, radicals of the structures xe2x80x94CHxe2x95x90CHxe2x80x94COOR5, xe2x80x94CH2CH2COOR5, xe2x80x94CH2CH,CN, xe2x80x94CH2CH2COCH3, xe2x80x94CH2COCH3, in which
R5 represents methyl or ethyl,
R6 represents hydrogen, amino, hydroxyl, methyl or halogen.
in the form of racemates, diastereomer mixtures or as enantiomerically pure or diastereomerically pure compounds, their pharmaceutically utilizable hydrates and/or salts, such as acid addition salts and the alkali metal, alkaline earth metal, silver and guanidinium salts of the carboxylic acids on which they are based, have a high antibacterial action against Helicobacter spp. and can be used for the eradication of this pathogenic bacterium.
Preferred compounds of the formula (I) are those in which
R1 represents tert-butyl which is optionally mono- or disubstituted by fluorine or cyclopropyl which is optionally substituted by 1 fluorine atom,
R2 represents hydrogen, alkyl having 1 to 4 carbon atoms or (5-methyl-2-oxo-1,3-dioxol-4-yl)-methyl,
A represents Cxe2x80x94R3, where
R3 represents hydrogen, fluorine, methoxy, difluoromethoxy, cyano alternatively, together with R1, can form a bridge of the structure xe2x80x94*xe2x80x94Oxe2x80x94CH2xe2x80x94CHxe2x80x94CH3 or xe2x80x94*Oxe2x80x94CH2xe2x80x94Nxe2x80x94CH3, where the atom marked by * is connected to the carbon atom of A,
R4 represents hydrogen, C1-C3-alkyl, radicals of the structures, xe2x80x94CH2CH2COOR5, xe2x80x94CH2CH2CN, xe2x80x94CH2COCH3, in which
R5 represents methyl or ethyl,
R6 represents hydrogen, amino or methyl,
and their pharmaceutically utilizable hydrates and/or salts, such as acid addition salts, and the alkali metal, alkaline earth metal, silver and guanidinium salts of the carboxylic acids on which they are based.
Particularly preferred compounds of the formula (I) are those in which
R1 represents tert-butyl which is optionally mono- or disubstituted by fluorine, or cyclopropyl,
R2 represents hydrogen, methyl or ethyl,
A represents Cxe2x80x94R3, where
R3 represents hydrogen, methoxy, difluoromethoxy, cyano alternatively, together with R1, can form a bridge of the structure xe2x80x94*Oxe2x80x94CH2xe2x80x94CHxe2x80x94CH3 or xe2x80x94*Oxe2x80x94CH2xe2x80x94Nxe2x80x94CH3, where the atom marked by * is connected to the carbon atom of A,
R4 represents hydrogen or methyl,
R6 represents hydrogen,
and their pharmaceutically utilizable hydrates and/or salts, such as acid addition salts, and the alkali metal, alkaline earth metal, silver and guanidinium salts of the carboxylic acids on which they are based.
The present invention also relates to the new compounds 8-cyano-1-cyclopropyl-6-fluoro-7-(2-oxa-5,8-diazabicyclo[4.3.0]non-8-yl)-1,4-dihydro4-oxo-3-quinolinecarboxylic acid and 1-cyclopropyl-8-difluoromethoxy-6-fluoro-1,4-dihydro-7-(2-oxa-5,8-diazabicyclo[4.3.0]non-8-yl)-4-oxo-3-quinolinecarboxylic acid, in particular in diastereomerically pure and enantiomerically pure form, and their pharmaceutically utilizable hydrates and/or salts, such as acid addition salts, and the alkali metal, alkaline earth metal, silver and guanidinium salts of the carboxylic acids on which they are based. 8-Cyano-1-cyclopropyl-6-fluoro-7-((1S,6S)-2-oxa-5,8-diazabicyclo[4.3.0]non-8-yl)-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid is particularly preferred.
The compounds which are suitable for the use according to the invention are in some cases already known from EP-A-0 350 733, EP-A-0 550 903 and from DE-A-4 329 600 or can be prepared by the processes described there.
If, for example, 9,10-difluoro-3,8-dimethyl-7-oxo-2,3-dihydro-7H-pyrido-[1,2,3-d,e][1,3,4]benzoxadiazine-6-carboxylic acid and 2-oxa-5,8-diazabicyclo-[4.3.0]nonane are used, the course of the reaction can be represented by the following equation: 
The 7-halogeno-quinolonecarboxylic acid derivatives employed for the preparation of the compounds of the formula (I) according to the invention are known or can be prepared by known methods. Thus 7-chloro8-cyano-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid and ethyl 7-chloro-8-cyano-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylate have been described in EP-A-0 276 700. The corresponding 7-fluoro derivatives can also be synthesized, for example, via the following reaction sequence: 
An alternative process for the preparation of the intermediate compound 2,4-dichloro-3-cyano-5-fluoro-benzoyl chloride, which serves as a starting material for the preparation of 7-chloro-8-cyano-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid (EP-A-0 276 700) and which can converted into 3-cyano-2,4,5-trifluoro-benzoyl fluoride, starts from 5-fluoro-1,3-xylene: 5-fluoro-1,3-xylene is dichlorinated in the nucleus under ionic conditions in the presence of a catalyst to give 2,4-dichloro-5-fluoro-1,3-dimethylbenzene and this is then chlorinated under free-radical conditions in the side chains to give 2,4-dichloro-5-fluoro-3-dichloromethyl-1-trichloromethylbenzene. This is hydrolysed via 2,4-dichloro-5-fluoro-3-dichloromethylbenzoic acid to give 2,4-dichloro-5-fluoro-3-formyl-benzoic acid and then reacted to give 2,4-dichloro-5-fluoro-3-N-hydroxyiminomethyl-benzoic acid. By treatment with thionyl chloride, 2,4-dichloro-3-cyano-5-fluoro-benzoyl chloride is obtained, which can additionally be reacted by means of a chlorine/fluorine exchange to give 3-cyano-2,4,5-trifluoro-benzoyl fluoride. 
The amines employed for the preparation of the compounds of the formula (I) according to the invention are known from EP-A-0 550 903, EP-A-0 551 653 and from DE-A-4 309 964.
An alternative to the synthesis of 1S,6S-2-oxa-5,8-diazabicyclo[4.3.0]nonane dihydrobromide or of the free base 1S,6S-2-oxa-5,8-diazabicyclo[4.3.0]nonane and the corresponding 1R,6R enantiomer is the following route:
The starting material for this synthesis is cis-1,4-dihydoxy-2-butene,which is reacted to give 1-tosylpyrrolidine after mesylation to the bis-mesylate with tosylamide. This is converted into the epoxide [lacuna] m-chloroperbenzoic acid. The ring-opening of the epoxide is carried out by heating with ethanolamine in isopropanol to give trans-3-hydroxy-4-(2-hydroxy-ethylamino)-1-(toluene-4-sulphonyl)-pyrrolidinein over 80% yield. The latter is then reacted with tosyl chloride in pyridine/tetrahydrofuran with cooling to give the tris-tosylate, which is cyclized under basic reaction conditions as a crude product mixed with some tetra-tosyl derivative to give racemic trans-5,8-bis-tosyl-2-oxa-5,6-diazabicyclo[4.3.0]nonane. At this stage, a chromatographic resolution is carried out with high selectivity on silica gel-bonded poly(N-methacryloyl-L-leucine-d-menthylamide) as the stationary phase. The desired enantiomer, (1S,6S)-5,8-bis-tosyl-2-oxa-5,6-diazabicyclo[4.3.0]nonane, is isolated with a purity of  greater than 99% ee. The removal of the p-tosyl protective groups is carried out using HBr-glacial acetic acid to give 1S,6S-2-oxa-5,8-diazabicyclo[4.3.0]nonanedihydrobromide, which can be converted into the free base using bases such as, for example, sodium or potassium hydroxide or with the aid of an ion exchanger. The analogous reaction sequence can also be used for the preparation of 1R,6R-2-oxa-5,8-diazabicyclo[4.3.0]nonane dihydrobromide. 
Synthesis of 1S,6S-2-oxa-5,8-diazabicyclo[4.3.0]nonane
Examples of compounds according to the invention which may be mentioned apart from the compounds mentioned in the preparation examples are the compounds mentioned in Table 1 below, which can be used both in racemic form and as enantiomerically pure or diastereomericallypure compounds.
The compounds according to the invention can crystallize in the form of their betaines or in the form of the salts having one to two mol of water.
The compounds according to the invention have a strongly antibiotic action and exhibit, together with low toxicity, a broad antibacterial spectrum against gram-positive and gram-negative microorganisms, above all, however, also against Helicobacter spp.
These valuable properties make possible their use as chemotherapeutic active compounds for the therapy of Helicobacter pylori infections and the gastroduodenal disorders associated therewith, which can be prevented, ameliorated and/or cured by the compounds according to the invention.
The compounds according to the invention can be used in various pharmaceutical preparations. Preferred pharmaceutical preparations which may be mentioned are tablets, coated tablets, capsules, pills, granules, solutions, suspensions and emulsions.
Although the compounds according to the invention are employed as monotherapeutic agents, if required they can also be used in combination with other therapeutics. By way of example, the following may be mentioned as combination components: nitroimidazole derivatives, for example metronidazole; proton pump inhibitors, for example omeprazole, pantoprazole or lansoprazole; H2 receptor antagonists, such as, for example, cimetidine, ranitidine, famotidine or nizatidine; bismuth compounds, such as, for example, bismuth salicylate or CBS (colloidal bismuth subcitrate); other antibiotics, such as, for example, amoxicillin, azlocillin or clarithromycin; antacids.
The minimal inhibitory concentrations (MIC), which are listed in Table 2 by way of example in comparison to ciprofloxacin for some of the compounds according to the invention, were determined in the agar dilution test on Columbia agar or Basis 2 agar (Oxoid) with 10% lysed horse blood either at pH 7 or pH 5 with 1 g/l of urea. The test substances were tested in replica dishes, which contained falling concentrations of the active compound at in each case doubled dilution. For inoculation, fresh Helicobacter cultures from liquid culture or suspension of the bacteria from agar plates were used. The inoculated agar plates were incubated at 37xc2x0 C. in an atmosphere containing 5-10% of CO2 for 48-72 hours. The MIC value (mg/l) which was read off indicates the lowest active compound concentration at which no growth was to be detected using the naked eye. The following Helicobacter isolates were used: H. felis ATCC 49179, H. pylori NCTC 1163 7, H. pylori clinical isolate 008.
For investigations in the animal model, female Swiss mice (8 to 12 weeks old, SPF breeding) were kept using commercially available feed and water. A defined H. felis strain (ATCC 49179) was used for colonization. The bacteria are administered by stomach tube as a suspension (0.1 ml containing 108-109 bacteria) 4 times in the course of 7 days. Alternatively to this, stomach homogenates of previously infected mice were used for infection.
3-5 days after establishment of the infection, the treatment with test preparations was begun. As a first treatment result, the bacterial reduction was determined as xe2x80x9cclearancexe2x80x9d 24 hours after the last treatment (for example 3, 7, 10, 14 days; 1-3 times daily). In some cases, the bacterial eradication was also determined 2-4 weeks after the end of treatment. Following the xe2x80x9cCLOxe2x80x9d test used in clinical diagnosis, a urease test on a microtitre basis was employed. Defined stomach biopsy samples were tested for colour change within 24 hours.
In Table 3, as an example of the surprisingly high in-vivo action of the compounds according to the invention, the therapeutic result after 7 days"" treatment of infected mice with 8-cyano-1-cyclopropyl-6-fluoro-7-((1S,6S)-2-oxa-5,8-diazabicyclo[4.3.0]-non-8-yl)-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid (Example 1A) and for 9-fluoro-3-methyl-10-((1S,6S)-2-oxa-5,8-diazabicyclo[4.3.0]non-8-yl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-d,e][1,3,4]benzoxadiazine-6-carboxylic acid (Example 2) is shown in comparison to treatment with ciprofloxacin: while with ciprofloxacin no clearance is achieved under these experimental conditions, in the compounds according to the invention this is 100%. A 10-day treatment of the mice with 2xc3x9710 mg/kg of 8-cyano-1-cyclopropyl-6-fluoro-7-((1S,6S)-2-oxa-5,8-diazabicyclo[4.3.0]non-8-yl)-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid even led to an eradication of the bacterium.